MicroRNA-34b/c suppresses uveal melanoma cell proliferation and migration through multiple targets

Abstract

Purpose: MicroRNAs (miRNAs) are endogenously expressed, small noncoding RNAs that inhibit gene expression by binding to target mRNAs. Recent studies have revealed that miRNAs function as tumor suppressors or oncogenes. In the present study, we investigated the role of miRNA-34b/c in uveal melanoma.

Methods: Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was performed to detect the expression level of miR-34b/c in uveal melanoma cells and primary samples. Subsequently, uveal melanoma cell proliferation was examined by the MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl] -2H-tetrazolium, inner salt) assay, clone formation assay, and flow cytometry. Cell apoptosis was measured by caspase3/7 assay. Cell migration was evaluated by transwell migration assay. The target of miR-34b/c was predicted by bioinformatics and validated by luciferase assay. In addition, the effect of miR-34b/c on c-Met, cell cycle-related proteins, v-akt murine thymoma viral oncogene homolog (Akt) and extracellular signal-regulated kinase (ERK) pathway was determined by western blotting.

Results: miR-34b/c expression, which was dramatically decreased in uveal melanoma cells and clinical samples, can be upregulated by doxorubicin and epigenetic drugs. The transfection of miR-34b/c into uveal melanoma cells leads to a significant reduction in cell growth and migration. miR-34b/c caused cell cycle G(1) arrest rather than the induction of apoptosis. Met proto-oncogene (c-Met) was identified as a target of miR-34b/c in uveal melanoma cells. Furthermore, miR-34b/c was confirmed to downregulate the expression of c-Met, p-Akt, and cell cycle-related proteins by western blotting.

Conclusions: Our results demonstrate that both miR-34b and miR-34c act as tumor suppressors in uveal melanoma cell proliferation and migration through the downregulation of multiple targets.

Figure 1

MicroRNA-34b/c expression was downregulated in human uveal melanoma cell line and clinical samples. A: Real time reverse transcriptase (RT)-PCR analysis was performed to detect the expression of miR-34b/c in clinical samples. The value for miR-34b/c in normal uveal tissue was set at 1, and the relative amounts of miR-34b/c in tumor tissues were shown as fold induction. Both miR-34b and miR-34c were dramatically decreased in five specimens as compared with normal tissues. N: normal uveal tissue; T: tumor tissue. B: The expression of miR-34b/c was measured by real time RT–PCR in uveal melanoma cell line SP6.5, as well as the primary uveal melanocytes. miR-34b/c was expressed in uveal melanocytes but dramatically decreased in uveal melanoma cells. U6 snRNA was used as an internal control.

Figure 2

MicroRNA-34b/c was upregulated by doxorubicin and epigenetic drugs. A: Real time reverse transcriptase (RT)-PCR analysis was performed to detect the expression of miR-34b/c in uveal melanoma cell line SP6.5, after treatment with DOX for 24 h and 48 h. B: SP6.5 cells were treated with 5-aza-dC at 1 μM or 5 μM alone, TSA (100 ng/ml) alone, or combinations of both. miR-34b/c expression level was determined by Real-time RT–PCR. The value for miR-34b/c in SP6.5 cells without any treatment was set at 1, and the relative amounts of miR-34b/c in cells treated with drugs were shown as fold induction. U6 snRNA was used as an internal control.

Figure 3

Ectopic microRNA-34 b/c inhibited SP6.5 cell proliferation. A: MTS cell proliferation assay was performed on days 1 to 5 as indicated after transfection into SP6.5 cells with either miR-34 b/c mimic or a negative control (NC). Results represent those obtained in three experiments. B: SP6.5 cells transfected with miR-34b/c or NC were seeded at low density. Colony formation was observed by staining with crystal violet after seven days. Typical results from three independent experiments are depicted. C: SP6.5 cells were transfected with miR-34b/c or NC. After 48 h, cells were collected, stained with propidium iodide, and analyzed by flow cytometry. The most representative results from three independent experiments are shown.

Figure 4

Transfection of microRNA-34 b/c did not induce cell apoptosis but did enhance cell sensitivity to DOX. Caspase 3/7 activity assay was performed on SP6.5 cells transfected with either miR-34b/c or a negative control (NC). Relative caspase 3/7 activity is indicated in comparison to the negative control. Cells were treated without (A) or with DOX (B). Results represent those obtained in three experiments.

Figure 5

Transfection of microRNA-34 b/c reduced uveal melanoma cell migration. Transwell migration assay of uveal melanoma cell lines was performed. SP6.5 cells were transfected with miR-34b/c or a negative control (NC) for 24 h and plated on cultured inserts in DMEM containing 20 ng/ml of hepatocyte growth factor (HGF) to assess the number of migrating cells. The number of cells that had migrated through the pores was quantified by counting 10 independent visual fields using a 20× microscope objective. *: Differences in cell migration between miR-34b/c and negative control transfected cells were significant, p<0.01.

Figure 6

c-Met was a target of microRNA-34 b/c. A: Two specific binding sites of miR-34b/c in the c-Met 3′ untranslated region (UTR) was marked with black color. Alignment between the predicted miR-34b/c target sites and miR-34b/c, the common 8 bp seed sequence for miR-34b/c:mRNA (mRNA) pairing is shown. B: Design of the pMIR luciferase reporter constructs, containing c-Met 3′ UTR, which was used to verify the putative miR-34b/c binding sites. C: SP6.5 cells were cotransfected with miR-34b/c, pLuc-MET 3′ UTR, and a pRL-SV40 reporter plasmid. The luciferase activity was measured after 24 h. Values are presented as relative luciferase activity after normalization to Renilla luciferase activity. *: Differences in luciferase activity between miR-34b/c and negative control transfected cells were significant, p<0.01.

Figure 7

MicroRNA-34 b/c downregulated c-Met, p-Akt and cell cycle proteins in uveal melanoma cells. SP6.5 cells were transfected with miR-34b/c or a negative control. Cell lysates were prepared and used for western blot analysis with c-Met, phosphorylated Akt (p-Akt), total Akt, phosphorylated ERK1/2 (p-ERK1/2), total ERK1/2, CDK4, CDK6, and phosphorylated-Rb (p-Rb) antibodies. Glyceraldehyde-3-phosphate dehydrogenase was used as a loading control. A: miR-34b/c downregulated the expression of c-Met and p-Akt. B: Cell cycle–related proteins CDK4, CDK6, and p-Rb were downregulated by miR-34b/c.